The invention relates to a method for assessing the genotoxicity of a compound liable to represent a risk to humans or animals.
At a time when combinatorial chemistry is developing, enabling the synthesis of countless novel compounds, short-term toxicological studies are occupying an increasingly important place in the context of the first steps of the development of these compounds, liable to be of value in industries such as the pharmaceutical, agronomic, food and cosmetics industries, etc.
Specifically, the development of a novel compound involves studies which are essential before the very first human exposure and which constitute the xe2x80x9cprerequisitesxe2x80x9d. These at least involve studies of toxicity by single and subacute administration and basal assessment of the mutagenic and genotoxic potential.
Since most carcinogenic substances are mutagenic, and vice versa, it is essential for genetic toxicology to use reliable tests which make it possible to come to safe conclusions regarding the potentially mutagenic effects of a compound, by gene mutations, chromosomal mutations or genomic mutations in somatic or germinal cells.
In order to assess the genotoxicity of the compounds of interest, industry makes use of tests which make it possible to demonstrate a clastogenic effect (breaking of chromosomes) or an aneugenic effect (spindle abnormality).
However, the authors of the present invention have shown that the demonstration of a clastogenic effect may be disturbed by compounds which are not clastogenic but which induce apoptosis with the presence of nuclear events. These tested compounds therefore emerge as xe2x80x9cfalse-positivesxe2x80x9d or xe2x80x9cexaggerated-positivesxe2x80x9d and are ruled out of any subsequent use.
The authors of the present invention have developed a method for assessing the genotoxicity of a compound, which makes it possible to be rid of this problem of xe2x80x9cfalse-positivesxe2x80x9d or xe2x80x9cexaggerated-positivesxe2x80x9d.
More precisely, a subject of the invention is a method for assessing the genotoxicity of a compound, in vitro, in which said compound is brought into contact with at least one cell or cell type overexpressing the bcl2 proto-oncogene and/or a related anti-apoptotic protein, and the genotoxic effects of said compound on said cell are observed.
In other words, a subject of the invention is the use of cells overexpressing the bcl2 proto-oncogene and/or a related anti-apoptotic protein, for assessing the genotoxicity of a compound by being free of the effects linked to apoptosis alone.
The compound to be tested may be any compound of natural or synthetic origin, which is designated indifferently xe2x80x9ccompoundxe2x80x9d, xe2x80x9cproductxe2x80x9d or xe2x80x9csubstancexe2x80x9d. It may be a mixture of several molecules, which may or may not be identified, such as, for example, an extract of animal or plant origin. The compound to be tested may be of therapeutic value, or may be useful in the chemical, agrochemical, food or cosmetics industry in particular.
According to a first embodiment of the invention, these positive genotoxic effects may be observed by the formation of a micronucleus or micronuclei.
This micronucleus test, described in particular by Matsuoka et al., 1993 and Kirsch-Volders et al., 1997, is based on the following principle:
During mammalian cell mitosis, chromosome fragments or whole chromosomes which have not undergone segregation will not be located in the main nucleus during telophase and may be observed, under the microscope, in the form of micronuclei separated from the main nucleus.
The DNA fragments which give rise to the micronuclei may be caused by either lesions to DNA (clastogenic or aneugenic effects of genotoxic compounds) or cleavage subsequent to apoptosis (effects of pro-apoptotic compounds).
According to a second embodiment of the invention, positive genotoxic effects of a compound may be observed by the presence of abnormalities of number and/or of structure of the chromosomes in metaphase.
This metaphase analysis test has, in particular, been described by Evans et al., 1987.
The principle of this test is as follows: the cells are treated with the compound to be tested, and then, by taking out and staining the cells blocked in metaphase (with a blocking agent such as colcemid), chromosomal abnormalities (breaks, rearrangements, etc.) are sought.
These two embodiments (micronucleus test and analytical test in metaphase) may advantageously be carried out in a complementary fashion.
They do not exclude other embodiments of the method of the invention, using cells overexpressing bcl2 and/or a related anti-apoptotic protein.
Moreover, since some compounds require metabolic activation in order to exert their genotoxic effects, it is possible to add, to the compound to be tested, a metabolic activator or activation system, such as xe2x80x9cS9 mixxe2x80x9d, containing a subcellular microsomal fraction of rat liver (Kirkland et al., 1989), in the method of the invention.
In accordance with the present invention, the compound to be tested is brought into contact with cells overexpressing the bcl2 proto-oncogene and/or a related anti-apoptotic protein. The overexpression of bcl2, known to be an inhibitor of apoptosis, creates an imbalance between inducers and inhibitors of apoptosis of the bcl2/bax family. The overexpression of the bcl2 protein thus prevents the cells from undergoing apoptosis.
The expression xe2x80x9cbcl2-related anti-apoptotic proteinxe2x80x9d is intended to mean any protein of the bcl2 family which has anti-apoptotic activity, the characteristics of this family being described by Biolo et al., 1999.
The homology between the various proteins of this family is concentrated in three regions, named BH1, BH2 and BH3, which control their abilities to dimerize with other members of the same family and also their apoptosis-regulating functions. All the anti-apoptotic members also contain a BH4 domain located close to their N-terminal end. These proteins also have, at their C-terminal end, hydrophobic amino acids which appear to be important for anchoring them in intracellular membranes.
Among the bcl2-related anti-apoptotic proteins, mention may preferentially be made of bcl-XL, which exhibits very strong homology with bcl2 (Biolo et al., 1999; Chao et al., 1995).
The cells used are eukaryotic cells, and preferentially mammalian cells. According to a preferred embodiment of the invention, they are CTLL-2 cells. CTLL-2cells, which are well known to those skilled in the art, originate from a continuous line of cytotoxic T lymphocytes which is a subclone derived from the C57bl/6 mouse. This line is available at the American Type Culture Collection under the number ATCC TIB-214 and has been described in a certain number of publications (Gillis et al., 1997; Hu et al., 1997). Other cell types may also be used, such as, for example, L5178Y, CHO, V79, fibroblasts or human or animal lymphoma cells, or other eukaryotic cells. A CTLL-2 line can be transfected with a plasmid containing bcl2 or a related sequence, according to standard transfection and transformation methods known to those skilled in the art. The preparing of CTLL-2-bcl2 cells has been described, in particular, in the article by Deng et al., 1993.
In accordance with the present invention, the cells may be transfected so as to overexpress a bcl2-related anti-apoptotic protein, such as bcl-XL, according to standard methods within the scope of those skilled in the art who are aware of the sequences of the corresponding genes (Bolse et al., 1993).
The method in accordance with the invention is efficient for detecting false-positives due to the apoptotic phenomenon. In addition, the method makes it possible to detect products which have a true clastogenic or aneugenic capacity without being an inducer of apoptosis.
Sensitivity and Reproducibility of the present method make it a valid method for assessing the genotoxicity of compounds on a large scale.